Although digitalis is highly toxic, it is still a very useful drug for treating heart failure. The inotropic action of heart muscle due to digitalis is thought to be caused by a rise in intracellular Na+ level resulting from inhibition of the NaK-pump. Recently, it has been demonstrated that palytoxin (PTX), an extremely potent marine toxin, vasoconstrictor, and tumor promoter, creates a new Na+ channel in venticular myocytes and enhances the cytoplasmic Na+ in cells. Now, using . NaK-ATPase proteoliposomes (PLs), we have obtained conclusive evidence that: (1) the NaK-pump is a cellular receptor for PTX; and (2) PTX in pM concentrations both induces the formation of a new Na+ channel and inhibits the pump. Since the PLs are prepared from a highly purified NaK-ATPase preparation and phospholipid-cholesterol liposomes, they can be used to simulate in vivo conditions with an in vitro system. Using PLs instead of living cells also avoids undesirable effects due to other cell components. From its extremely high potency and functional similarities to digitalis, we are confident that studying the PTX-NaK-pump interaction in the PLs is well worth the effort. We intend to independently characterize the two PTX effects, i.e., channel formation and inhibition. In this proposal, therefore, we plan to carry out the following studies: (a) the resolution of the difference in PTX sensitivity between proteoliposomal and fragmental NaK-ATPase; (b) the characterization of the PTX-induced channel; (c) the determination of which NaK-pump conformer interacts with PTX directly; and (d) the PTX binding location on the NaK-pump molecule. These approaches should help us to determine the structure-activity relationship of PTX. Since PTX is a huge organic compound (MW 2680) with many possible derivatives, our knowledge about the structure-activity relationship of PTX to the NaK-pump will help us to discover new vasoconstriction agents and cardiotonic drugs. We also believe that the general knowledge that can be obtained by studying such an extremely potent vasoconstrictor will enable researchers to understand how PTX and similar compounds induce the constriction of heart muscle.